Synergistic anti-inflammatory and antioxidant dietary supplement compositions

ABSTRACT

Disclosed herein are novel dietary supplement compositions comprising enriched 3-O-acetyl-11-keto-β-boswellic acid and enriched demethylated curcuminoids, wherein the composition exhibits anti-inflammatory, antiulcerogenic and anti-oxidant activities. Also disclosed are novel compositions comprising enriched 3-O-acetyl-11-keto-β-boswellic acid, enriched demethylated curcuminoids that have a synergistic effect on specific inhibition of—COX-2 and 5-LOX activity and optionally containing glucosamine, resveratrol, garlic extract, chondroitin, methlysulphonymethane, bromelain, serratiopeptidase, quercitine, gallic acid, caffeic acid, green tea extract, aspirin and ibuprofen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of parent U.S. application Ser. No.12/593,581 filed on Sep. 8, 2009, which is a U.S. national stageapplication based on International Application PCT/IN2007/000135,published as WO 2008/120220. The entire disclosure of each priorapplication is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to synergistic compositions for curing orpreventing lipoxygenase mediated and free radical mediated disorders inmammals and various forms of degenerative musculoskeletal diseases suchas rheumatoid arthritis and osteoarthritis. The present inventionfurther relates to compositions exhibiting synergistic inhibition of theexpression and/or activity of inducible cyclooxygenase-2 (COX-2) and5-lipoxygenase (5-LOX). Particularly, the compositions comprise of anenriched 3-O-acetyl-11-keto-β-boswellic acid (AKBA), enricheddemethylated curcuminoids and optionally containing other ingredients,which shows a synergistic anti-inflammatory and antioxidant property.

BACKGROUND OF THE INVENTION

inflammatory diseases affect more than fifty million Americans. As aresult of basic research in molecular and cellular immunology over thelast ten to fifteen years, approaches to diagnosing, treating andpreventing these immunologically-based diseases has been dramaticallyaltered. One example of this is the discovery of an inducible form ofthe cyclooxygenase enzyme. Constitutive cyclooxygenase (COX), firstpurified in 1976 and cloned in 1988, functions in the synthesis ofprostaglandins (PGs) from arachidonic acid (AA). Three years after itspurification, an inducible enzyme with COX activity was identified andgiven the name COX-2, while constitutive COX was termed COX-1.

COX-2 gene expression is under the control of pro-inflammatory cytokinesand growth factors. Thus, the inference is that COX-2 functions in bothinflammation and control of cell growth. While COX-2 is inducible inmany tissues, it is present constitutively in the brain and spinal cord,where it may function in nerve transmission for pain and fever. The twoisoforms of COX are nearly identical in structure but have importantdifferences in substrate and inhibitor selectivity and in theirintracellular locations. Protective PGs, which preserve the integrity ofthe stomach lining and maintain normal renal function in a compromisedkidney, are synthesized by COX-1. On the other hand, PGs synthesized byCOX-2 in immune cells are central to the inflammatory process. Thediscovery of COX-2 has made possible the design of new drugs andsynergistic compositions that reduce inflammation without removing theprotective PGs in the stomach and kidney made by COX-1.

Leukotriens and 5(s)-HETE are important mediators for inflammatory,allergic and obstructive process. Leukotriens increases micro vascularpermeability and are potent chemo tactic agent. Inhibition of5-lipoxygenase indirectly reduces the expression of TNF-α.5-Lipoxygenase is therefore the target enzyme for identifyinginhibitors, which have potential to cope with a variety of inflammationand hypersensitivity-based human diseases including asthma, arthritis,bowel diseases such as ulcerative colitis and circulatory disorders suchas shock and ischemia.

Free radicals play a major role in the initiation and progression of awide range of pathological diseases like cancer, Alzheimer's,Parkinson's and cardiovascular disease. In the food industry, freeradicals have been found to be responsible in the deterioration of foodsduring processing and storage. In view of this, considerable attentionhas been given to the addition of antioxidants in foods andsupplementation of antioxidants to biological systems to scavenge freeradicals.

A yellow pigmented fraction isolated from the rhizomes of Curcuma longacontains curcuminoids belonging to the dicinnamoyl methane group.Curcuminoids are present to the extent of 3 to 5 percent in the rawmaterial. They are considered the most important active ingredients andare believed to be responsible for the biological activity of Curcumalonga. Though their major activity is anti-inflammatory, curcuminoidshave been reported to possess antioxidant, antiallergic, wound healing,antispasmodic, antibacterial, antifungal, antitumor and antiHIV activityas well. Curcumin was isolated in 1815 and structurally defined in 1910.Other major curcuminoids isolated from Curcuma longa includedemethoxycurcumin and bisdemethoxycurcumin. Curcuminoids may be found inother botanicals in addition to Curcuma longa, such as Curcumaxanthorrhiza and Curcuma zedoaria. Cureuminoids are well known for theiranti-inflammatory activity. Turmeric is one of the oldestanti-inflammatory drugs used in Ayurvedic medicine.

The pharmacokinetics involving the safety, toxicity, dose range andbiological properties of turmeric and its components, including curcuminis known, and the turmeric is readily available in various food stores.

The anti-inflammatory properties of curcumin were shown to inhibit the5-lipoxygenase activity in rat peritoneal neutrophils as well as the12-lipoxygenase and the cyclooxygenase activities in human platelets(Ammon, H. P. T. et al., J. Ethopharmacol., 1993, 38, 113-119). Curcuminhad no significant effect on quercetin-induced nuclear DNA damage, lipidperoxidation and protein degradation and thus has the unique potentialof acting as both pro- and antioxidants, depending on the redox state oftheir biological environment (Saura, C. et al., Cancer Letters, 1992,63, 237-241).

Among the bibliographic references of the most interesting patentsexisting in the data banks, application FR2,655,054 about the cellularprotection agents contained in curcuminoids obtained from Curcuma longa,ascorbic acid and/or dismutase super oxide (DSO) stands out.Curcuminoids have a known antioxidizing effect, as well as ascorbic acidand DSO that carry out a synergic effect.

The gum resin of the plant Boswellia serrata (Burseraceae) has long beenin use for the treatment of rheumatoid arthritis and gout by thepractitioners of Ayurvedic medicines in the Indian system of medicine.Various extracts of the gum resin have shown potent anti-inflammatoryand anti-arthritic activity in laboratory animals, as well as duringclinical trials (Atal, C. K. et al., Ind. J. Pharm., 1980, 12, 59;Pachnanda, V. K. et al., Ind. J. Pharm., 1981, 13, 63). In a detailedpharmacological study, Singh et al have established that the alcoholicextract of B. serrata gum resin displayed marked anti-inflammatoryactivity in carrageenan induced edema in rats and mice and dextranoedema in rats (Singh, G. B. et al., Agents and Action, 1986, 18, 407).It has also been recognized in the past that the ethanolic extract ofthe gum resin of B. serrata inhibits the formation of Leukotriene B4 inrat peritoneal neurophils. Leukotriene B4 is one of the importantmediators of inflammatory reactions (Ammon, H. P. T. et al., PlantaMedica, 1991, 57, 203).

A composition of enriched demethylated curcuminoids, enriched3-O-acetyl-11-keto-β-boswellic acid (AKBA) and optionally otheringredients for preserving the health of joint tissues, for treatingarthritis or other inflammatory conditions with COX-2 and 5-LOXspecificity has not yet been discovered. A composition comprisingdemethylated curcuminoids, AKBA and optionally other ingredients tosynergistically inhibit COX-2 and 5-LOX with high specificity andsupport the normalization of joint function has also not yet beendiscovered.

An important need therefore exists for dietary composition suitable forhuman patients that are synergistic in that they have stronger effectsthan the sum of the effects of the individual components and alsosynergistic with standard clinical treatment of inflammatory conditionsand in the prior art, there is no such synergistic 5-lipoxygenaseinhibitory composition for inflammatory diseases.

It is therefore an objective of the present invention to provide anon-toxic dietary supplement composition, which prevents or cure5-lipoxygenase mediated disorders like inflammatory diseases (e.g.rheumatoid arthritis, osteoarthritis, and periodontal disease), asthama,and bowel disease such as ulcerative colitis and circulatory disorderssuch as shock and ischemia

It is a further objective of the present invention to provide acomposition for preventing free radical mediated disorders such ascancer, Alzheimer's, Parkinson's and cardiovascular disease.

Thus, it would be useful to identify an enriched natural compositionthat would specifically inhibit or prevent the synthesis of COX-2 and5-LOX. Such a composition, containing enriched demethylatedcurcuminoids, enriched AKBA, which would be useful for preserving thehealth of joint tissues, for treating arthritis or other inflammatoryconditions, asthma, bowel disease such as ulcerative colitis andcirculatory disorders such as shock and ischemia has not previously beendiscovered.

U.S. Pat. No. 6,521,271 described the methods of promoting improvementof skin condition by administering a turmeric component and glycolicacid to a patient afflicted with a skin disorder.

U.S. Pat. No. 6,264,995 described an herbal composition for reducinginflammation in bones and joints by inhibiting the enzymecyclooxygenase-2. This composition is prepared from holy basil;turmeric, ginger, green tea, rosemary, huzhang, Chinese goldthread,barberry, oregano and scutellariae baicalensis.

U.S. Pat. No. 6,841,177 disclosed antiproliferative andphotosensitization activities of Curcuma longa extract and its use inproliferative diseases such as psoriasis, as reducers of plasmaticfibrinogen and the Apolipoprotein B/Apolipoprotein A-I quotient, withoutaltering other coagulation parameters.

U.S. Pat. No. 6,440,468 disclosed a method for obtaining apolar andpolar extracts of Curcuma and applications thereof. A process forobtaining the apolar extract comprises: (a) extracting the rhizomes withan organic solvent; (b) filtration and evaporation to dryness of theextract; (c) dissolution of the oleoresin obtained in hot conditions,precipitation while allowing to cool down and filtration of the solid;(d) drying and recrystallizing the solid in order to obtain a producthaving a purity in curcuminoids higher than 90%. A process for obtainingthe polar extract comprises: (i) extraction of the rhizomes with waterat 50-70° C. and (ii) filtration and evaporation of the water.Application of the compositions and preparations as catchers of freeradicals and anti-ageing agents, as well as reducing agents to reducethe plasma levels of lipid peroxides in human beings are disclosed.

U.S. Pat. No. 5,494,668 disclosed a method of treating degenerativemusculoskeletal diseases such as rheumatoid arthritis and osteoarthritisin an animal, typically a human, comprises administering to the animal,typically in a convenient dosage form, a therapeutically effectiveamount of the beneficiated extracts of the plants ashwagandha (Withaniasomnifera), sallai guggul (Boswellia serrata), turmeric (Curcuma longa),and ginger (Zingiber officinale) in a predetermined proportion relativeto each other with or without other biologically active inorganicingredients, such as zinc sulphate. The beneficiated plant extracts aremade in accordance with a novel process which is also disclosed.

U.S. Pat. No. 5,629,351 disclosed a novel fraction comprising a mixtureof boswellic acids, wherein the fraction exhibits anti-inflammatory andantiulcerogenic activities. Also disclosed is a novel boswellic acidcompound exhibiting anti-inflammatory, antiarthritic and antiulcerogenicactivities. Also disclosed is a process for isolating a boswellic acidfraction and individual boswellic acids therefrom.

US Patent Application 20030152585A1 disclosed herbal compositioncomprising a mixture of herbs such as Tinospora cordifolia, Aloe Vera,Curcuma loraga, Withania somnifera, Achyranthus asperea, Ocinum sanctumand Picorrhiza kurroa for treatment of hematological malignancies.

U.S. Pat. No. 6,979,470 disclosed curcuminoid compositions comprising aneffective amount of a curcuminoid species and an effective amount of aditerpene lactone species, a triterpene species or derivatives thereofthat have a synergistic effect on specific inhibition of inducible COX-2activity and have minimal effect on COX-1 activity.

US Patent Application 20040247700A1 disclosed curcuminoid compositionsexhibiting synergistic inhibition of the expression and/or activity ofcyclooxygenase-2 and this composition is useful for treating e.g.inflammation or arthritis, comprising curcuminoid and diterpene lactoneor triterpene, and specifically inhibits inducible cyclooxygenase-2(COX-2) activity.

US Patent Application 20030096027A1 and 20050129791A1 disclosedcurcuminoid compositions exhibiting synergistic inhibition of theexpression and/or activity of cyclooxygenase. It is also disclosed thiscomposition for treating e.g. inflammation or inflammation baseddiseases, comprising curcuminoid species and alpha-acid (hops plantproducts) or beta-acid (lupulones).

US Patent Application 20030108628A1 described curcuminoid compositions,which exhibits synergistic inhibition of the expression and/or activityof cyclooxygenase. It also disclosed this composition for treating e.g.inflammation or arthritis, comprising curcuminoid and diterpene Intoneor triterpene, and specifically inhibits inducible cyclooxygenase-2(COX-2) activity.

US Patent Application 20050191375A1 described synergisticanti-inflammatory pharmaceutical compositions and related methods usingcurcuminoids or methylxanthines. It also disclosed the composition forreducing inflammation or treating e.g. pain, cancer, rheumatoidarthritis, psoriasis, ulcerative colitis and conjunctivitis comprisesfraction isolated or derived from hops and methylxanthine.

US Patent Application 20050123632A1 described anti-inflammatory activityof a specific turmeric extract. It also disclosed mixture of turmericoils for pharmaceutical or nutraceutical composition for treatinginflammation e.g. rheumatoid arthritis, comprises hexane solublefraction.

US Patent Application 20030216600A1 described novel polyhydroxycurcumins having antioxidant activity and these new polyhydroxycurcuminsare useful as antioxidants.

European Patent 1133992A1 described novel pharmacological activities ofCurcuma longa extracts. The use of aqueous alcoholic extract of Curcumalonga in composition having e.g. photosensitizing, antiproliferative andfibrinogen level reducing activity, used e.g. for treating psoriasis isalso disclosed.

The patent WO03007975A1 described curcuminoid compositions, whichexhibits synergistic inhibition of the expression and/or activity ofcyclooxygenase-2. It also disclosed the composition useful for treatinge.g. inflammation or arthritis, comprising curcuminoid and diterpenelactone or triterpene, and specifically inhibits induciblecyclooxygenase-2 (COX-2) activity.

The patent WO06062681A1 described curcuminoid compositions exhibitingsynergistic inhibition of the expression and/or activity ofcyclooxygenase-2. The formulation comprises, as a first component aneffective amount of a curcuminoid species and an effective amount of asecond component selected from the group consisting of an alpha-acidspecies (hops plant products) or beta-acid species (lupulones) orderivatives thereof.

The patent WO05084230A2 described synergistic anti-inflammatorypharmaceutical compositions and related methods using curcuminoids ormethylxanthines. The composition useful for reducing inflammation ortreating e.g. pain, cancer, rheumatoid arthritis, psoriasis, ulcerativecolitis and conjunctivitis comprises fraction isolated or derived fromhops and methylxanthine is disclosed.

The patent GB2388539A1 disclosed two polyherbal formulations fortreating cancer, comprises mixture of six or four herbs, or mixture ofactive ingredients extracted or synthesized from herbs. The firstformulation comprises a mixture of Glycine max, Lycopersicon esculentum,Allium sativum, Curcuma longa, Linum usitatissimum and Convolvulusarvensis. The second formulation comprises a mixture of Tinosporacordifolia, Withania somnifera, Phyllanthus emblica and Asparagusracemosus.

U.S. Pat. No. 5,629,351 described boswellic acid compositions andpreparation thereof. The composition comprises a new fraction containingknown boswellic acids and new 2-alpha, 3-alpha-dihydroxyurs-12-en-24-oic acid is useful as synergistic anti-inflammatory,antiarthritic and antiulcerogenic agent.

The patent AU0075253A5 described novel pharmacological activities ofCurcuma longa extracts. The use of aqueous alcoholic extract of Curcumalonga in composition having e.g. photosensitizing, antiproliferative andfibrinogen level reducing activity, used e.g. for treating psoriasis isalso disclosed.

US patent application 20040166178A1 described3-O-acetyl-11-keto-boswellic acid or its extract in a topicalcomposition for treatment and prevention of lines and to relax the skin.The patent also disclosed the use of composition comprising Boswelliaserrata extract for topical application to the skin, as an agent tosoften lines and/or relax the skin.

The patent WO0057893A1 disclosed the composition comprises an extract ofBoswellia plant and carrier for use on hair and skin for reducingirritation.

The patent WO0062751A2 disclosed the composition comprises Boswelliaplant extract, carrier and flavor ingredient for treating irritation orinflammation in mouth.

The patent WO03077860A2 disclosed boswellia composition comprises anenriched 3-β-acetyl-11-keto-β-boswellic acid (AKBA) for treating e.g.inflammation. It also disclosed a manufacturing process byperacetylating a boswellia serrata pentacyclic terpene acidic fractionextract to provide 3-beta-acetoxy-11-keto-beta-boswellic acid.

The patent IN0182615A described a process for the isolation of a newboswellic acid from the gum resin of the plant boswellia serrata. Italso disclosed the composition for use on hair and skin for reducingirritation comprises extract of Boswellia plant and carrier.

U.S. Pat. No. 6,589,516 described compositions containing Boswelliaextracts. The Composition for use on hair and skin for reducingirritation comprises extract of Boswellia plant and carrier is alsodisclosed.

Thus none of the prior art mentioned above relates to a compositioncomprising enriched AKBA extracts and enriched demethylated curcuminoidsshow synergistic 5-lipoxygenase inhibition.

It is therefore an object of the present invention to provide anon-toxic dietary supplement composition, which prevents or cure COX-2and 5-lipoxygenase mediated disorders, inflammatory diseases likerheumatoid arthritis, periodontal disease, asthma, bowel disease such asulcerative colitis and circulatory disorders such as shock and ischemia,free radical mediated disorders such as cancer, Alzheimer's, Parkinson'sand cardiovascular disease.

SUMMARY OF THE INVENTION

The present invention provides a novel synergistic dietary supplementcomposition for preventing lipoxygenase mediated and free radicalmediated disorders.

The present invention provides a dietary synergistic mixture of enriched3-O-acetyl 11-keto-β-boswellic acid (AKBA) from Boswellia serrata, andenriched demethylated curcuminoids from Curcuma longa.

The present invention further provides a dietary synergistic mixture ofenriched AKBA from Boswellia serrata, and enriched demethylatedcurcuminoids from Curcuma longa and optionally containing one or more ofglucosamine, resveratrol, garlic extract, chondroitin,methlysulphonylmethane, bromelain, Serratiopeptidase and quercitine.

The present invention further provides a composition for preventing orcuring inflammatory disease like rheumatoid arthritis, osteoarthritis,periodontal disease, asthma, bowel disease such as ulcerative colitisand circulatory disorder such as shock and ischemia.

The present invention further provides a composition for preventing freeradical mediated disorders such as cancer, Alzheimer's, Parkinson's, andcardiovascular diseases.

The present invention provides a dietary synergistic mixture of enrichedAKBA extract from Boswellia serrata and enriched demethylatedcurcuminoids from Curcuma longa wherein the said extract from Boswelliaserrata is enriched upto 100% of AKBA.

The present invention provides a dietary synergistic mixture of enrichedAKBA from Boswellia serrata, and enriched demethylated curcuminoidextract from Curcuma longa wherein the said extract from Curcuma longais enriched upto 100% of demethylated curcuminoids.

The present invention provides a dietary synergistic mixture of enrichedAKBA from Boswellia serrata, and enriched demethylated curcuminoids fromCurcuma longa wherein the said composition functions synergistically toinhibit the inducibility and/or activity of 5-LOX and COX-2.

In our co-pending application US2004073060 dated on Apr. 15, 2004 wehave disclosed a process for enriching AKBA in the range of 30-95% fromBoswellia serrata extract. The enriched AKBA extract is several timesmore potent 5-Lipoxygenase inhibitor compared to the commerciallyavailable Boswellia extracts. A detailed study on the structuralrequirements for boswellic acids indicated that, of all the six acids,3-O-acetyl-11-keto-β-boswellic acid, hereinafter referenced as AKBA(FIG. 1) shows most pronounced inhibitory activity against 5-LOX.

5-Loxin is the brand name for a Boswellia extract having not less than30% of AKBA and this is used to prepare the composition and the extracthaving 2-95% of AKBA could also be used.

In our co-pending application PCT/IN05/00337 dated on Oct. 13, 2005, wehave disclosed a process for enriching demethylated curcuminoids upto100%. The enriched detnethylated curcuminoids is a potent 5-lipoxygenaseand free radical inhibitor. The components present in the enricheddemethylated curcuminoids are shown in FIG. 2. The demethylated Curcumalonga extract having 15-100% of total demethylated curcuminoids (formula1-4) is used in this composition.

The components present in enriched demethylated curcuminoids are allnatural products. The compounds (formula 1, 3 and 4) are present inCurcuma longa as minor natural products (Mimura, A. et al., U.S. Pat.No. 5,266,344 and Jiang, H. et al., J. Chromatography A, 2006, 1111,21-31). The compound (formula 2) is isolated from Curcuma domestica(Nakayama, R. et al., Phytochemistry, 1993, 33, 501-502).

Further, the scientists at National Cancer Institute screenedcurcuminoids and demethylated curcuminoids for anti-HIV activity. Theyfound that the demethylated curcuminoid (formula 1) inhibited HIV-1integrase with IC₅₀ value below 10 μM.

The mixture having 15-100% of total demethylated curcuminoids (formula1-4) is used in this composition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for reducinginflammation. In particular, the invention provides a composition havingsynergistic activity comprising enriched 3-O-acetyl 11-keto-β-boswellicacid (AKBA) derived from Boswellia serrata, and enriched demethylatedcurcuminoids derived from Curcuma longa. The invention provides AKBAenriched Boswellia serrata extracts in combination with enricheddemethylated curcuminoids for use in treating inflammation in a patientprophylactically and/or therapeutically.

The invention is also directed to compositions comprising enriched AKBAfrom Boswellia serrata and enriched demethylated curcuminoids fromCurcuma longa and optionally containing one or more of glucosamine,resveratrol, garlic extract, chondroitin, methlysulphonymethane andbromelain, serratiopeptidase, and quercitine.

Non-steroidal anti-inflammatory drugs (NSAID's) are most commonly usedremedies for rheumatic disease. But regular use of NSAID's increases thedevelopment of ulcers in the stomach and duodenum. Hence, there has beena tremendous surge in demand for natural NSAID's because of theirestablished safety and efficacy, through decades of usage by variouscultures.

The present inventive composition is composed of natural extracts andproven safety from decades of use in traditional system of medicine.Besides, Boswellia serrata extract is known to display antiulcerogenicactivity.

In a preferred embodiment, the composition of the invention comprisesenriched AKBA and enriched demethylated curcuminoids, and optionallycontaining glucosamine, resveratrol, garlic extract, chondroitin,methlysulphonymethane and bromelain, serratiopeptidase, and quercitine.

In a preferred embodiment of the invention, the composition of theinvention comprises 2-95% of 3-O-acetyl-11-keto-β-boswellic acid.

In a more preferred embodiment of the present invention, the enrichedboswellia extract contains 30% AKBA, commercially known as 5-Loxin®.

The synergistic new dietary supplement composition comprising enrichedAKBA and enriched demethylated curcuminoids is in the ratio of 9:1 to1:9.

In a preferred embodiment of the invention, the composition comprisesenriched 3-O-acetyl-11-keto-β-boswellic acid (AKBA) and enricheddemethylated curcuminoids in the ration of 5:1 to 1:1 (w/w) showssynergistic 5-lipoxygenase inhibitory activity (Table 1).

The said dietary supplement composition optionally contains one or moreof glucosamine, resveratrol, garlic extract, ginger extract,chondroitin, methysulfonylmethane and bromelain, serratiopeptidase, andquercitine.

The said glucosamine is selected from glucosamine hydrochloride orglucosamine sulphate or N-acetlyglucosamine.

The composition for dietary oral supplementation use comprising 10-90%of enriched AKBA extract and 90-10% of enriched demethylatedcurcuminoids.

The composition for dietary oral supplementation use comprising 30-50%of enriched AKBA extract, 10-20% of enriched demethylated curcuminoidsand 40-50% glucosamine hydrochloride.

The said dietary supplement composition contains 30-40% of enriched AKBAextract, 5-20% of enriched demethylated curcuminoids, 50-60% ofglucosamine hydrochloride and 5-10% of resveratrol.

The composition for dietary oral supplementation use comprising 30-60%of enriched AKBA extract, 5-20% of enriched demethylated curcuminoidsand 5-20% of resveratrol.

The composition for dietary oral supplementation use comprising 30-60%of enriched AKBA extract, 5-20% of enriched demethylated curcuminoidsand 10-30% of methylsulfonylmethane.

The composition for dietary oral supplementation use comprising 30-60%of enriched AKBA extract, 5-20% of enriched demethylated curcuminoidsand 10-30% of ginger extract.

The composition for dietary supplementation use comprising 20-30% ofenriched AKBA extract, 5-10% of enriched demethylated curcuminoids,40-50% of glucosamine hydrochloride, 5-10% of resveratrol, 10-15% ofgarlic extract and 10-15% of ginger extract.

The composition comprising 20-30% of enriched AKBA extract, 5-10% ofenriched demethylated curcuminoids, 30-40% of glucosamine hydrochloride,5-10% of chondroitin sulfate, 10-15% of methylsulfonylmethane and 5-10%of bromelain is for dietary supplementation use.

The invention also provides compositions having good antioxidantactivity for pharmaceutical or dietary/nutraceutical or cosmetic use,characterized by comprising, enriched AKBA, enriched demethylatedcurcuminoids, gallic acid, eaffeic acid and green tea extract.

The said dietary supplement composition contains 30-50% of enriched AKBAextract, 5-10% of enriched demethylated curcuminoids, 5-10% of gallicacid, 5-10% of caffeic acid and 20-30% of green tea extract.

Further, the invention relates to the composition comprising enrichedAKBA from Boswellia serrata, and enriched demethylated curcuminoidsderived from Curcuma longa, and optionally containing one or more ofnon-steroidal anti-inflammatory agents like aspirin (acetyl salicylicacid), ibuprofen [α-methyl-4-(2-methylpropyl)benzeneacetic acid], etc.

The said dietary supplement composition contains 30-50% of enriched AKBAextract, 5-20% of enriched demethylated curcuminoids and 10-30% ofaspirin.

The said dietary supplement composition contains 30-50% of enriched AKBAextract, 5-20% of enriched demethylated curcuminoids and 10-30% ofibuprofen.

The said synergistic dietary supplement composition comprising enrichedAKBA and enriched demethylated curcuminoids and optionally containingother ingredients is found to show good antioxidant activity (Table 2).

A method of treating a subject suffering from an inflammatory disease,wherein said inflammatory disease result from biomolecules secreted fromactivated and degranulated mast cells, said inflammatory disease beingselected from the group consisting of osteoarthritis, cancer,fibromyalgia, atherosclerosis, inflammatory bowel disease, interstitialcystitis, irritable bowel syndrome, asthama, migraines, angina, chronicprostatitis, eczema, arthritis, multiple sclerosis, psoriasis, sun burn,and periodontal disease, comprising the step of administering to saidsubject an effective amount (e.g., an amount effective to treat, slowthe progression of, etc) of said composition.

A method of preventing a subject suffering from free radical mediateddisease, wherein said free radical mediated disease result from theradicals generated in the body, said free radical disease being selectedfrom cancer, Alzheimer's, Parkinson's, and cardiovascular diseasescomprising the step of administering to said subject an effective amountof the said composition.

Active new dietary supplement composition of the present invention maybe produced by the procedures described herein or variations thereof,which will apparent to those skilled in the art.

A further aspect of the present invention describes a pharmaceuticalformulation comprising a composition along with in a pharmaceuticallyacceptable carrier (eg, an aqueous or a non aqueous carrier).

Preferred embodiments relating to the different compositions of thesubject invention are illustrated below:

Composition 1:

A dietary supplement composition was prepared by mixing unit doses ofthe following components:

Enriched AKBA extract 5 g Enriched demethylated curcuminoids 1 g

Composition 2:

A dietary supplement composition was prepared by mixing unit doses ofthe following components:

Enriched AKBA extract 5 g Enriched demethylated curcuminoids 1 g

Composition 3:

A dietary supplement composition was prepared by mixing unit doses ofthe following components:

Enriched AKBA extract 2 g Enriched demethylated curcuminoids 2 g

Composition 4:

A dietary supplement composition was prepared by mixing unit doses ofthe following components:

Enriched AKBA extract   2 g Enriched demethylated curcuminoids 0.5 gGlucosamine hydrochloride 2.5 g

Composition 5:

A dietary supplement composition was prepared by mixing unit doses ofthe following components:

Enriched AKBA extract  1.5 g Enriched demethylated curcuminoids 0.25 gGlucosamine hydrochloride   3 g Resveratrol 0.25 g

Composition 6:

A dietary supplement composition was prepared by mixing unit doses ofthe following components:

Enriched AKBA extract 1.5 g Enriched demethylated curcuminoids 0.25 gGlucosamine hydrochloride 2 g Resveratrol 0.25 g Garlic extract 1 g

Composition 7:

A dietary supplement composition was prepared by mixing unit doses ofthe following components:

Enriched AKBA extract  1.5 g Enriched demethylated curcuminoids 0.25 gGlucosamine hydrochloride   2 g Resveratrol 0.25 g Garlic extract 0.75 gGinger extract 0.75 g

Composition 8:

A dietary supplement composition was prepared by mixing unit doses ofthe following components:

Enriched AKBA extract 2.5 g Enriched demethylated curcuminoids 0.5 gGallic acid 0.5 g Caffeic acid 0.5 g Green tea extract   1 g

The invention is further explained with the help of the followingexamples:

EXAMPLE 1 Synergestic 5-lipoxygenase Inhibitory Activity

The new dietary supplement compositions are screened for their5-lipoxygenase inhibitory potential using colorimetric method. The assaymixture contained 50 mM phosphate buffer (pH 6.3), 5-lipoxygenase,various concentrations of test composition (5 μg, 10 μg, 20 μg) indimethylsulphoxide and linolenic acid in a total volume of 0.5 mL, after5 min incubation of above reaction mixture 0.5 mL ferric xylenol orangereagent is added and OD is measured after two minutes at 585 nm usingspectrophotometer. Controls are run along with test in a similar mannerexcept using vehicle instead of test substance solution. Percentinhibition is calculated by comparing absorbance of test solution withthat of control.

The results are illustrated in the following table

TABLE 1 5-Lipoxygenase inhibitory activity % of inhibition S. No. Nameof the compound 5 μg 10 μg 20 μg 1 Composition 1 22.4 34.1 49.7 2Composition 2 29.3 39.9 49.2 3 Composition 3 44.5 57.5 64.14 4Composition 4 19.4 27.3 45.1 5 Composition 5 9.1 14.9 34.9 6 Composition6 15.6 22.7 36.5 7 Composition 7 17.1 24.1 44.5 8 Composition 8 12.525.6 53.2 9 Enriched AKBA extract 0 12.5 32.7 10 Enriched demethylated33.9 62.9 67.5 curcuminoids The higher the % inhibition values, thehigher the activity.

From the above 5-lipoxygenase inhibitory values (Table 1), it clearlyshows the synergism of the compositions. For example, at 5 μgconcentration, the enriched demethylated curcuminoids and enriched AKBAextract exhibited 33.9% and 0% inhibition respectively. Whereas thecomposition 3, which contains 1:1 ratio of enriched demethylatedcurcuminoids and enriched AKBA extract exhibited 44.5% inhibition. Thisinhibitory value of the composition is higher than the individualcomponents.

EXAMPLE 2 Antioxidant Activity of the Compositions

The dietary supplement compositions are screened for their antioxidantactivity by superoxide free radical-scavenging method. The superoxidefree radical-scavenging activity is determined by the NBT (nitro bluetetrazolium) method. The reaction mixture contained EDTA (6.6 mM), NaCN(3 μg), riboflavin (2 μM), NBT (50 μM), various concentrations of thetest compositions in ethanol and a phosphate buffer (58 mM, pH 7.8) in afinal volume of 3 ml. Optical density is measured at 560 nm. The testtubes are uniformly illuminated with an incandescent lamp for 15 min,after which the optical density is measured again at 560 nm. Thepercentage inhibition and superoxide radical generation is measured bycomparing the absorbance values of the control and those of the testcompounds.

The dietary supplement compositions are screened for their antioxidantactivity by DPPH method. DPPH (1,1-diphenyl-2-picrylhydrazyl) radicalscavenging activity is measured based on the reduction of methanolicsolution of the colored DPPH. Free radical scavenging ability of thetest compositions in ethanol added to the methanolic solution of DPPH isinversely proportional to the difference in initial and final absorptionof DPPH solution at 516 nm. The reaction mixture contained 1×10⁻⁴ mMmethanolic solution of DPPH and various concentrations of test drugs.The percentage inhibition is determined by comparing the absorbancevalues of test and control tubes.

The results are illustrated in the following table:

TABLE 2 Antioxidant activity Superoxide (NBT) DPPH S. No. Name of thecompound IC₅₀ μg IC₅₀ μg 1 Composition 1 26.0 2.88 2 Composition 2 16.52.62 3 Composition 3 7.5 1.33 4 Composition 4 50.0 13.06 5 Composition 573.0 28.75 6 Composition 6 79.6 26.85 7 Composition 7 74.0 27.43 8Composition 8 8.75 4.64 9 Enriched AKBA extract >100 — 10 Enricheddemethylated 2.8 — curcuminoids The lower the IC₅₀ values, the higherthe antioxidant activity.

The above antioxidant activity data (Table 2) reveled that all thecompositions exhibited good antioxidant activity. Further, thesecompositions are potent antioxidant agents in comparison withcommercially available antioxidants like Vitamin E etc.

EXAMPLE 3 Synergestic Anti-Inflammatory Activity of Composition-1

Test animals (rats, 200-300 g) of either sex were randomly distributedinto different groups, with 5 animals in each group. Rats weresupplemented daily with either demethylated curcuminoids (50 mg/kg) orenriched AKBA extract (50 mg/kg) or composition 1 (50 mg/kg) in 1% CMCfor 28 days. The control group received same volume of vehicle (1% CMC),a standard drug, prednisolone (10 mg/kg, orally) was used in the controltreatment group. On the day 14, 100 μl of Freund's Complete Adjuvant(FCA) was injected subcutaneously in the sub planter region of the lefthind paw. Blood samples were collected from each animal on the previousday of FCA challenge and at the day of termination of experiment i.e.14days after challenge. At the 28^(th) day paw volumes were measured andafter sacrificing the animals, liver tissues were excised and stored at−80° C. until further use. The pro-inflammatory markers like nitrite,IL-1β in sera, and paw edema were measured.

EXAMPLE 4 Synergestic Anti-Inflammatory Activity of Composition 7

Test animals (rats, 200-300 g) of either sex were randomly distributedinto different groups, with 5 animals in each group. Rats weresupplemented daily with either demethylated curcuminoids (50 mg/kg) orenriched AKBA extract (50 mg/kg) or glucosamine (50 mg/kg), orreseveratrol (50 mg/kg), garlic extract (50 mg/kg) or ginger extract (50mg/kg), or composition 7 (50 mg/kg) in 1% CMC for 28 days. The controlgroup received same volume of vehicle (1% CMC); a standard drug,Prednisolone (10 mg/kg, orally) was used in the control treatment group.On the day 14, 100 μl of Freund's Complete Adjuvant (FCA) was injectedsubcutaneously in the sub planter region of the left hind paw. Bloodsamples were collected from each animal on the previous day of FCAchallenge and at the day of termination of experiment i.e. 14 days afterchallenge. At the 28^(th) day paw volumes were measured and aftersacrificing the animals, liver tissues were excised and stored at −80°C. until further use.

The percent of inhibitions are as follows:

Demethylated curcuminoids 42.3% Enriched AKBA extract 36.5% Glucosamine5.5% Reseveratrol 20.5% Garlic extract 25.2% Ginger extract 20.2%Composition 7 49.5%

DESCRIPTION OF DRAWINGS

The results are summarized in FIGS. 1, 2 and 3 for IL-1β, nitrite andedema volume respectively.

FIG. 1 depicts bar diagrammatic representation of modulation of serumIL-1b concentration (pg/ml) in different groups of animals on the13^(th) day (before FCA challenge) and on the 28^(th) day (14 days afterchallenge) of experiment. 1, CMC control; 2, Prednisolone; 3,Curcuminoids 95% (50 mg/kg body weight); 4, demethylated curcuminoids(10 mg/kg); 5, enriched AKBA extract (50 mg/kg); 6, a mixture of naturalcurcuminoids and enriched AKBA extract (50 mg/kg); 7, Composition 1 (50mg/kg). Each bar represents mean±SD obtained from 5 animals in eachgroup.

FIG. 2 shows bar diagrammatic representation of modulation of serumnitrite concentration (mM) in different groups of animals on the 13^(th)day (before FCA challenge) and on the 28^(th) day (14 days afterchallenge) of experiment. 1, CMC control; 2, Prednisolone; 3,Curcuminoids 95% (50 mg/kg body weight); 4, demethylated curcuminoids(10 mg/kg); 5, enriched AKBA extract (50 mg/kg); 6, a mixture of naturalcurcuminoids and enriched AKBA extract (50 mg/kg); 7, composition 1 (50mg/kg). Each bar represents mean±SD obtained from 5 animals in eachgroup.

FIG. 3 shows bar diagrammatic representation of percent inhibition ofpaw edema after 14 days of inoculation of Freund complete adjuvantinoculation in sub planter region of the left hind paw in animals ofdifferent groups. 1, Prednisolone; 2, Curcuminoids 95% (50 mg/kg bodyweight); 3, demethylated curcuminoids (10 mg/kg); 4, enriched AKBAextract (50 mg/kg); 5, a mixture of natural curcuminoids and enrichedAKBA extract (50 mg/kg); 6, composition 1 (50 mg/kg). Each barrepresents the mean value in percent inhibition with respect to CMCcontrol group.

It will be evident to those skilled in the art that the invention is notlimited to the details of the foregoing illustrative example and thatthe present invention may be embodied in other specific forms withoutdeparting from the essential attributes thereof, and it is thereforedesired that the present embodiments and examples be considered in allrespects as illustrative and not restrictive, reference being made tothe appended claims, rather than to the foregoing description, and allchanges which come within the meaning and range of equivalency of theclaims are therefore intended to be embraced therein.

We claim:
 1. A method of treating a free radical mediated disease,wherein said free mediated disease results from radicals generated inthe body, said free radical mediated disease being selected from cancer,Alzheimer's, Parkinson's and cardiovascular disease, comprising the stepof: administering to said subject an effective amount of a compositioncomprising enriched 3-O-acetyl-11-keto-β-boswellic acid (AKBA) fromBoswellia serrata extract and enriched demethylated curcuminoids fromCurcuma longa extract; said enriched demethylated curcuminoidscomprising from 15% by weight to about 100% by weight of at least onecompound selected from the group consisting of compounds of Formula I,Formula 2, Formula 3 and Formula 4:


2. A method of treating a free radical mediated disease in a subjectresulted from radicals generated in the body comprising the step ofadministering to said subject an effective amount of a compositioncomprising enriched 3-O-acetyl-11-keto-β-boswellic acid (AKBA) fromBoswellia serrata extract and enriched demethylated curcuminoids fromCurcuma longa extract; said enriched demethylated curcuminoidscomprising at least one compound selected from the group consisting ofcompounds of Formula I, Formula 2, Formula 3, and optionally comprisinga compound of Formula 4:


3. The method of claim 2, wherein said enriched demethylatedcurcuminoids comprise from 15% by weight to about 100% by weight of atleast one compound selected from the group consisting of compounds ofFormula I, Formula 2, and Formula 3.